During the current reporting period we have focused on the following projects: 1) Studies of a New Autoinflammatory Disease Caused by Mutations in RIPK1 Members of three families presented with a previously undescribed autoinflammatory disorder characterized by early-onset periodic fever episodes, severe intermittent lymphadenopathy, organomegaly, and abdominal pain. In some individuals this disease was selectively responsive to therapeutic IL-6 inhibition with tocilizumab. Exome sequencing revealed that RIPK1 was the only gene in which a variant from all three families satisfied filtering criteria. In one family the sole affected individual had a de novo heterozygous mutation, p.Asp324Asn, the second family demonstrated 3-generation inheritance of the p.Asp324His heterozygous mutation in 5 affected individuals, and the third family had a de novo heterozygous mutation, p.Asp324Tyr, in the sole affected individual. These three mutations result in different substitutions at the same critical residue that is specifically required for RIPK1 cleavage by caspase-8. This aspartate is highly conserved across species, and all three mutations are absent from human variant databases. The mutations impaired RIPK1 cleavage by caspase-8 as well as by caspase-6 in vitro, though cleavage in human cells appeared to be mediated predominantly by caspase-8. We designate this clinical condition 'cleavage-resistant RIPK1-induced autoinflammatory (CRIA)' syndrome. To define the mechanism for this disease, we established a collaboration with John Silke's laboratory at the Walter and Eliza Hall Institute in Melbourne, Australia to generate appropriate mouse models. It should be noted that residue 325 in the mouse sequence is homologous to residue 324 in humans. Whereas Ripk1-/- mice die postnatally from systemic inflammation, Ripk1 D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by combined loss of Casp8 and Ripk3 but not by loss of Ripk3 or Mlkl alone. In addition, loss of RIPK1 kinase activity also prevented Ripk1 D325A/D325A embryonic lethality, however the mice died before weaning from multi organ inflammation in a RIPK3 dependent manner. Consistently, Ripk1 D325A/D325A and Ripk1 D325A/+ cells were hypersensitive to RIPK3 dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1 D325A/+ mice were viable, but hyper-responsive to inflammatory stimuli in vivo. These results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but maintains inflammatory homeostasis throughout life. A manuscript describing these findings has been accepted for publication in Nature. 2) Neutrophil Biology in PAPA Syndrome Pyogenic arthritis with pyoderma gangrenosum and acne (PAPA syndrome) is caused by mutations in PSTPIP1, which encodes a pyrin-binding protein. Patients develop severe neutrophil-mediated inflammation of the skin and joints. To study the role of neutrophils in PAPA, we provided samples from 12 PAPA patients to NIAMS collaborators expert in assessing neutrophil function. Their studies demonstrated that circulating low density granulocytes (LDGs) are elevated in PAPA subjects. PAPA sera exhibit impaired neutrophil extracellular trap (NET) degradation and this is corrected with exogenous DNase1. Recombinant human IL-1beta induces NET formation in PAPA neutrophils but not healthy control neutrophils. NET formation in healthy control neutrophils is induced by PAPA serum and this effect is inhibited by the IL-1 receptor antagonist, anakinra. NETs from PAPA neutrophils and LDGs stimulate IL-6 release from healthy macrophages. NETs are detected in skin biopsies of patients with PAPA syndrome in association with increased tissue IL-1beta, IL-8, and IL-17. Furthermore, LDG gene signatures are detected in PAPA skin. These data underscore the importance of neutrophil NET formation in the pathophysiology of PAPA syndrome, and suggest new therapeutic strategies for this severe and often treatment-refractory condition. A manuscript describing the results of this study was published during the current reporting period in the Annals of the Rheumatic Diseases.